The principal objective of these studies is to chemically characterize some of the proteins involved in the biosynthesis, degradation and function of cyclic AMP. The approach is that of isolating, purifying and characterizing the proteins involved in both the regulation of cyclic AMP levels by cells and the regulation of cell function by cyclic AMP, utilizing appropriate cellular models for each aspect of the study. A continuing analysis of soluble and membrane- bound protein kinase will be made in an effort to understand the precise molecular mechanisms by which kinase activity is regulated. The possibility that the enzyme from cardiac muscle may exist in two forms, phospho- and dephospho, cyclic AMP-activable and cyclic AMP-inactivable, respectively, is being explored. The role of cyclic nucleotide binding proteins in regulating the distribution of free and bound cyclic nucleotide will be analyzed in eukaryotes and in a prokaryotic model system in order to determine whether any aberrancies exist in neoplastic cells. The quaternary structure of beef heart phosphodiesterase is under investigation. We also propose to continue the investigations on insulin-secreting islet cells in vitro. Once cell lines are fully established, mutants defective in insulin synthesis, processing or secretion will be developed and used to ascertain the mechanism by which cyclic AMP mediates cellular secretory processes. The protein kinases, phosphodiesterases and adenylate cyclases of these cells are being studied to provide the basis for characterizing mutants defective in one or more of these enzymes. Microorganisms will be used to analyze the regulation of cylic AMP turnover under conditions of rapid metabolic change. Selected microorganisms will also be used to study the regulation of adenylate cyclase activity by two hormones: glucagon and insulin. Genetic tools will be employed to analyze the interaction of these hormones with their receptors and with adenylate cyclase in an effort to understand their physiological effects.